Glucans, particularly beta (1-3)-glucans, have been very extensively studied, and have been shown to have a variety of pharmacological activities, including but not limited to anti-cholesterolaemic activity, hypoglycaemic activity, and stimulation of the immune system. For this reason beta-glucan comprising products have been used as feed additives in poultry, animal, fish or crustacean production. Glucans are insoluble polymers. The present invention reveals novel yeast derived materials comprising increased amounts of hydrolysed water soluble yeast carbohydrates and other soluble molecules.
The cell wall of Saccharomyces cerevisiae is primarily composed of beta-linked glucan, which is mainly a backbone of beta(1-3)-linked glucose units, with a minor component of inter and intra molecular branching via beta (1-6)-linkages. The yeast glucans in the prior art are mostly water insoluble polymers. The present invention reveals novel yeast derived materials comprising increased amounts of various water soluble hydrolysed yeast carbohydrates including specific polymer material comprising β6-linked glucose as major structure, and soluble α-mannose materials and other soluble molecules with specific molecular size.
The yeast derived material of prior art have been described to comprise mannans insoluble glycoproteins or possible as oligosaccharides. The present invention revealed novel α-mannose materials with specific molecular size mainly in low molecular weight polysaccharide range. The soluble α-mannose materials are in a preferred embodiment major carbohydrate component. The novel fractions further have useful characteristics including clearness, reduced taste and odor, especially when the yeast is produced from spent yeast from beer production.
Because of the very wide use of yeasts in the food and brewing industry, as well as in the production of industrial-grade alcohol, spent yeast cells are a major industrial by-product. Yeast-derived products themselves have considerable commercial value, for example in such products as yeast extracts, flavouring agents, flavour potentiators such as guanosine monophosphate and inosine monophosphate, and in the manufacture of enzymes, fine chemicals and products for use in the biochemical and pharmaceutical industries, such as trehalose, thymidine, nucleosides and nucleotides, etc. Waste yeast from the brewing industry is a major source of beta-glucans.
In addition, other species of yeast are also useful as a source of beta-glucans or mannose containing glycans, including but not limited to other yeast strains of Saccharomyces cerevisiae, other yeast using food or beverage fermentation processes such as other Saccharomyces species including e.g. Saccharomyces carlsbergiensis, Kluyveromyces fragilis, and Candida strains such as Candida utilis. All of these yeast strains can be produced using culture in food grade nutrients either by batch fermentation or continuous fermentation.
The purification of beta-glucans from yeast and other organisms has been extensively investigated, and a variety of methods is known. Most of these rely on the insolubility of beta (1-3)-glucan in alkali or in organic solvents. The principal known methods are:
(a) High temperature extraction with concentrated sodium hydroxide, followed by high temperature extraction with acid and precipitation with ethanol (e.g. Manners, D. J. et al., Biochem. J. 135 19-30 (1973), Jamas, S. et al., U.S. Pat. Nos. 4,810,646, 5,028,703, and 5,250,436). Many of these protocols require preliminary homogenisation of the yeast cells, and many require multiple repetition of each extraction step.(b) Extraction with concentrated sodium hydroxide, followed by high temperature acid extraction and enzyme treatment to modify or purify the glucan (see for example Czech Patent Application No. 890038 by Masler, L. et al. which reports purification of beta-D-glucan by alkali-acid extraction, followed by treatment with enzymes having amylase activity).(c) Extraction of yeast cell wall preparations resulting from autolysis or enzyme degradation of yeast with concentrated phenol:water (1:1) (see for example U.S. Pat. No. 4,138,479 by Truscheit, E. et al.).(d) Extraction with organic solvents such as isopropanol, ethanol, acetone, or methanol either alone or in the presence of alkali (see for example Japanese Patent publications No. 7051081, 6340701, 5295003, and 3002202; European Patent Application No. 515216).
Acid treatment is known to reduce the number of beta (1-6)-linkages in the glucan material and this results in an increase in viscosity.
The cell wall of yeast is mainly composed of:
(i) fibrillar, alkali insoluble beta (1-3)-linked glucan, with side branches of beta (1-6)-linked glucan.
(ii) alkali-soluble beta (1-3)-linked glucan with side branches of beta (1-6)-linked glucan.
(iii) amorphous acid-soluble beta (1-6)-glucan, with intermittent beta (1-3)-linkages.
(iv) amorphous alkali-soluble mannan linked to proteins.
Existing methods to isolate the beta-glucans commonly use a multi-step alkali-acid extraction process. The alkali extraction steps remove most of the amorphous mannoprotein and glucan material, and the subsequent acid extraction steps remove the glycogen and most of the beta (1-6)-side branches from the fibrillar predominantly beta (1-3) linked glucan. A final solvent extraction step is sometimes used to remove lipids.
The glucan methods involve strong alkaline extraction and optionally weak acid treatment to obtain insoluble mainly beta3-linked glucans. The present invention includes more effective acid hydrolysis preferably in elevated temperatures to produce more soluble material and an alkaline hydrolysis to improve the solubilization, the alkaline reaction is used to extract alkali soluble material but the soluble fraction is not discarded but retained. The present invention further comprise separation and/or fractionation steps to remove insoluble material likely including β3-glucan, and optionally recycling the insoluble material to hydrolysis step to produce more soluble carbohydrates, and to remove low molecular weight materials such as monosaccharides or low molecular weight oligosaccharides or practically all oligosaccharides and removing salts. It is realized that acid and alkaline treatments produce salts.
It is clear that, given the retail price of glucan for some applications, the cost of producing glucan using existing published or patented methods is not commercially viable. These methods have at least one major disadvantage, they are aimed at only producing the fibrillar, alkali-insoluble form of glucan. Other forms of glucan and the mannan present in the cell wall are removed as by-products of the process. These represent an additional amount of glucan, which could have significantly increased the glucan yield, and which may be functionally important. In addition, after or between hydrolysis treatments soluble components are discarded whereas insoluble, mostly beta(1-3)glucan is retained. The method of production of present invention can be used to produce a soluble β6-glucose linkage structure comprising carbohydrate or saccharide materials for, used in or as immunostimulatory and/or modulatory compositions. The increased solubility and the reduction of the amount of β3-glucans structures distinguishes present soluble materials from known β-glucose containing materials.
Mannan is a polymer composed of mannose units. In yeasts, mannan is associated with protein in both the external surface of the yeast cell wall, as a muscigenous polysaccharide, and in the inner cell membrane. It generally accounts for about 20-50% of the dry weight of the cell wall. Mannan is linked to a core-peptide chain as an oligomer or polymer. The complex contains about 5-50% proteins. Oligomeric mannan is bonded directly to serine and threonine, whereas polymeric mannan is bonded to asparagine via N-acetylglucosamine. In the manno-protein complex, the mannose units are linked by .alpha.-1,6, .alpha.-1,2 and .alpha.-1,3-linkages. The present invention produced concentrated soluble mannans and mannose glycans, which have useful biological activities and distinct characteristics in NMR analysis.
Mannan-oligosaccharides (MOS) have suggested to be released from yeast cell walls by proteolytic action. Proteolysis produced mannan may have high molecular weight, contain allergenic proteins and likely have large molecular weight and poor solubility. Such released MOS type products have limited usefulness. In a specific embodiment the present method may include additional enzymatic and proteolytic steps. It is realized that the present methods the alkaline hydrolysis beta-eliminates O-linked and/or N-linked mannose glycopeptides. It further realized that the peptide materials may have negative effects of the product structure and even have negative immunological.
More soluble and useful mannans according to the present invention effectively bind to bacterial pathogens of the intestinal tract and block their ability to colonize the intestinal tract. For example, E. coli, Salmonella spp. and Vibrio cholera have proteins on their surface (lectins) which bind to the mannose sugar residues of the mannans. The present invention revealed novel active saccharide compositions comprising majority of glycans with molecular weight larger than oligosaccharide and capable of polyvalent and oligovalent presentation of oligosaccharide epitopes. The present method further produced high amounts of useful terminal oligosaccharide sequences for pathogen binding. The usefulness of oligovalent and polyvalent carbohydrate epitopes for anti-adhesion therapies and treatments against infections or for interactions with cell surface lectin receptors is well-known in the art and frequently reviewed e.g. by K-A. Karlsson and in Nathan Sharon's publication since Beachey 1981 (Beachey, E. H. (1981) J. Infect. Dis. 143, 325-345). The polyvalent glycans are used for anti-adhesion for preventing the binding of pathogens to patients tissue. By definition oligosaccharide contains less than 10 monosaccharide residues, Mw less than 1700, while present material comprise major amount of carbohydrates larger than about 5000 Da. The present materials are useful against the pathogens and harmful microbes because of the high valency improving binding and solubility of the novel mannose glycans.
There is a clear need in the art for a rapid and inexpensive method of saccharide fraction extraction, which avoids loss of alkali soluble glucans and mannans, which has improved recovery of glucans and mannans and which results in a biologically active preparation.
We have now surprisingly found that soluble immunostimulatory composition that contains saccharide fraction can be isolated using a simple two-hydrolysis steps involving procedure and that excellent yields of a soluble product with high immunostimulatory activity is obtained. Hydrolysis may be supplemented by treatment with enzymes or other agents or mechanical, pneumatic or hydrostatic treatment or with heat and if desired, the properties of the saccharide fraction can be modified by acid treatment, degree of homogenisation or by varying the type of enzyme used.